DESCRIPTION (From the applicant's abstract): Afferent input supports many neuronal functions in vivo including neuronal survival in the developing brain. Imbalance of the process of neuronal survival and apoptosis is linked to a growing list of development- and aging related neurological abnormalities characterized by neuronal death including Alzheimer's and Huntington's diseases. Neuronal activity-dependent cell survival is mediated by calcium influx. But the process by which calcium influx promotes neuronal survival remains largely unknown. The long-term objective of our research is to understand the mechanisms by which neuronal activity regulates gene expression. This present application is specifically designed to study how membrane depolarization-induced calcium influx affects the regulation and function of the transcription factor, myocyte enhancer factor 2 (MEF2), in the context of a cell survival model of cultured primary neonatal cerebellar granule neurons. The specific aims of this application include: 1. Identify the kinases and phosphatases that regulate MEF2 activity in calcium-dependent neuronal survival; 2. determine calciium-regulated sites of phosphorylation and dephosphorylation on MEF2A; 3. characterize the mechanisms by which MEF2 mediates calcium-dependent neuronal survival. Membrane depolarizing concentrations of extracellular potassium chloride mimics neuronal activity in vivo to promote survival of neonatal cerebellar granule neurons. This model will be used to determine by kinase assay or phospho Westernblot analysis the activities of specific isoforms of p38 MAPK, PKCs as well as calcineurin in response to calcium influx. The critical sites of (de)phosphorylation on MEF2A by these kinases and phosphatase will be determined by a combined approach of phosphopeptide mapping and phosphoamino acid analysis. Dimerization, subcellular localization, DNA binding, and transcription activation assays, combined with mutagenesis and phospho-antibody analysis, will be used to determine the effects of (de)phosphorylation of these sites on MEF2 function following calcium signals. Finally, the mechanisms by which MEF2 mediates survival will be studied by establishing whether KC1 and MEF2 regulate the expression of pro-survival gene bc1-2 in bc1-2 promoter-driven luciferase reporter assay and whether Bc1-2 can rescue neurons from apoptosis when calcium-dependent activation of MEF2 is blocked. These experiments will define the mechanisms of calcium-dependent, MEF2-mediated neuronal survival which are essential to our understanding of both physiological and pathological changes in developing neurons.